JPH04507094A - Feline T cell lymphotropic lentivirus polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the invention of feline T cell lymphotropic lentivirus polypeptide This application is filed under U.S. Patent Application No. 348,784 by O'Connor et al. This is a partial continuation application for “Feline T Cell Lymphtropic Lentivirus”). U.S. Patent Application No. 348,784 to O'Connor et al. U.S. Patent Application No. 293,906 (Application filed on January 5, 1989 1 Name of the invention: “Cat-Neel Lymph Tropic Wrench” This is a continuation-in-part application for a monoclonal antibody against viruses. Also, this US Patent Application No. 293,906 is a U.S. Patent Application No. 279,989 (1 Application filed on 1285/988 1 Name: “Feline T cell lymphotropic 1 nontivirus” This is a continuation-in-part application for a monoclonal antibody against cancer. And these outputs The entire application, including drawings, is incorporated herein.

This invention is based on feline T-cell lymphotropic (a tendency to change lymph). The present invention relates to the purification of polypeptides from antivirus (hereinafter referred to as FIV).

FIV is a veterinary virus originally isolated from cats that exhibits an AIDS-like syndrome. (Pederson et al. r235Science 790.1987) rus is a member of the same group as human immunodeficiency virus (HIV), which causes AIDS in humans. belongs to the group. Bederson et al. Detection of antibodies is described. Also, 0.1M NaCl and 1rnM Sucrose gradient in Tris (Trts) base at pH 7.4 containing EDTA ( Virus purified by centrifugation on Sucrose gradients) Antibodies to FIV can also be detected by Western blotting using It is said that Here, several protein bands (polymer layers) are detected. However, no antigenic comparisons were performed. The positions of these bands correspond to the major core proteins. (major core protein) There is. Here, the main core proteins are p24 gag precursor protein, p55 ° and endonuclease protein, p32. These are about HIV. Ru.

Hederson et al., U.S. Patent Application No. 1.987, filed August 26, 1987; "Lentivirus" (this is different from the preceding virus of this application) The above-mentioned results are described in In addition, individuals infected with FIV Major proteins obtained by Western blotting of cell lysates. The band is approximately 22-26kD, usually about 24kD; 50-60kD, usually about 55kD: and 28-36kDS4 is said to have about 32kD.

Summary of the invention First, the present invention provides an epitope (antigenic determinant, complex protein) of FIV antigenic polypeptide. Purified polypeptides containing each of the basic constituent elements that form the structure of the antigen molecule. Featuring petite. This polypeptide may be glycosylated or not. You don't have to. Antigenic polypeptides can generate antibody responses in cats means a polypeptide. The polypeptide is a polypeptide of at least 5 amino acids. It may be a peptide fragment (fragment), or it may be in a FIV particle (particle). It may also be a naturally occurring polypeptide. Fragme Agents can also be obtained from naturally occurring polypeptides. For example, naturally occurring poly Peptides may also be enzymatically digested. It can also be purified by separation or the desired Synthesis of genetically encoded polypeptides and expression of polypeptides in desired expression systems It may also be achieved by expression of lipeptides. This expression system can be used, for example, in bacteria or enzymes. Mother (yeast) or mammalian expression system.

Here, epitope refers to a single antigenic site of an antigenic polypeptide.

Such epitopes are particularly suitable for monochrome antigenic polypeptides of FIV. It can be recognized by monoc1ona1 antibody.

Purification also refers to, for example, other cellular constituents that are mixed with naturally occurring FIV polypeptides. means to separate this polypeptide from.

Preferably, the polypeptide is used for preparing monoclonal antibodies against it. It is desired that it be sufficiently pure to be able to be used for. In addition, polyp is sufficiently pure that the amino acid sequence of the putide can be determined using standard techniques This is desirable. Generally, a purified polypeptide is biologically active . This makes it suitable for the preparation of monoclonal antibodies, and it is suitable for the preparation of monoclonal antibodies. Suitable for the detection of naturally occurring antibodies in liquid Cse.m).

According to a preferred embodiment, the purified polypeptide is FIV gag- or a polypeptide fragment of at least 20 amino acids obtained from the nv polypeptide; have at least 75% amino acid homology to the even more preferable Alternatively, the purified polypeptide will contain all gag or env polypeptides. and amino acid sequences with at least 75% homology. Furthermore, purification 26, gp40. Includes gploo and gp130.

Next, the present invention provides a method for detecting antibodies against FIV in a sample, comprising: providing a purified polypeptide; Bring them into contact under conditions that will cause this to happen. That is, the antibody in the sample and the purified formation of antibody/polypeptide complexes between polypeptides, and Detect body formation. The presence of this antibody/polypeptide complex indicates that F Indicates the presence of IV antibodies.

Third, the present invention uses 50 nucleotides naturally occurring in FIV particles. A sequence of 50 nucleotides with at least 90% homology to the characterized by a prepared nucleic acid having a Here, purification refers to polypeptides and other The target nucleic acid can be substantially separated from all naturally occurring substances such as means. Preferably, the nucleic acid is completely separated from materials such as those mentioned above and is pure. Nucleic acids become a solution or are held in cells that do not occur naturally.

i.e. bacterial cells, other viral particles, and non-feline eukaryotic cells. Where is it held? 90% homology means that at least 45 of the 50 nucleotides are means a match to the target nucleotide sequence.

According to a preferred embodiment, the nucleic acid of interest is an epitope of an antigenic polypeptide. , that is, a polypeptide containing an epitope of the FIV gag or e/nv polypeptide. Encode the peptide. More preferably, the nucleic acid is carried out by an expression vector. transported and expressed in bacteria, fungi, and other eukaryotic cells, such as mammalian cells be able to.

In the context of the present invention, purified polypeptides may include one or more of the following: Contains contiguous amino acids from the beginning to the end. This The sequences are expressed using the standard notation for amino acids as follows:

(1) l/-Q-! 1i-R-G-3-G-F-V-C-F-N-C-ni-ni -P-G-H-L-A-R-Q-3-Ho r(2) F-1-Q-T-V-N -G-V-P-Q-Y-V-A-L-D-P-ni-M-V-8o r (3) 5 -V-Q-8-R-G-Q-G-P-V-A-P-N.

Applicants provide polypeptides suitable for detecting FIV antibodies, thereby This made it possible to accurately detect FIV infection. In addition, FIV in cats have provided polypeptides useful in the production of vaccines to prevent diseases caused by.

Other features and usefulness of the invention will become apparent from the description of the embodiments and claims.

DESCRIPTION OF THE PREFERRED EMBODIMENT First, I will briefly explain the drawings.

drawing Figure 1 shows polyacrylamide gel electrophoresis (PAGE) and Commassie Confirmed by staining with Blue R250 (lane A) (identif ied) This is a photograph of the main virus associated protein of FIV, and the molecular weight The standard is shown in lane B.

Figure 2 shows the cases in which the ELISA test for FIV antibodies confirmed positive results. Western immunotherapy of FIV antibodies found in serum from It is a photograph of a plot (western immunoblot) analysis.

Figure 3 graphically represents the elution of FIV polypeptide during HPLC purification. It is something that

Figure 4 is a photograph of an SDS polyacrylamide gel after electrophoresis and staining with silver. , indicating the purity of various FIV polypeptides.

Figure 5 shows the nucleic acid sequences at various sites of the cloned FiV nucleic acid. The corresponding amino acid sequence is shown.

antigenic FIV polypeptide A general description of FIV polypeptide antigens useful in this invention is provided above.

Polypeptides useful in this invention include viruses isolated as follows. It can be purified from Purified polypeptides can also be processed by enzymatic treatment or other Isolated by standard methods. Furthermore, this polypeptide - Can be synthesized by an in vitro expression system. child In the expression system, the DNA encoding for the FIV polypeptide is cloned and expressed in bacterial, yeast, or mammalian cell expression systems .

Such NA can be isolated and expressed as described below. this Polypeptides can also be synthesized by standard chemical methods, e.g. Polypeptide segments of each FIV polypeptide are synthesized as follows. be able to. In this example, the FIV polypeptide is directly obtained from FIV infected cells. in the form of naturally occurring polypeptides in FIV virus particles. It was. The present invention is not limited to this example, but the polypeptide of this invention including a variety of alternative methods recognized by those skilled in the art. molecule of this polypeptide In terms of quantity, a 30kD polypeptide refers to p30, and this weight of glycoprotein Ting is gp30.

The master seed virus used for culture was FIV, which was isolated from $2427. were obtained from a continuous cat cell line infected by Here, #2427 FIV is CRFK-FjV or Petaluma 5trafn, Dr. Nails Fuedaso, University of California, Davis, California This is due to the The parental cell line is a clone that has been persistently infected with FIV. These are Nder cat kidney cells. This cell line was published on July 13, 1988. It has been deposited in the Lycantype Culture Collection, and its deposit number is C. It is RL9761. Applicants and their designated agents are aware that the term of this patent is Before finishing, make sure to feed this again if this culture dies. do. This may be more than 5 years or 30 years after the final requirement for culture. be responsible even if and to inform about the place of deposit for the issuance of such patents. It is at such times that the deposit is ultimately made available to the public. .

Previously, deposits were made under the guidance of 37 CFR 1-14 and 35 U.S.C. 112. Deposit is available to the Commissioner of Patents pursuant to the provisions.

Other virus cultures are described by Felderson et al. Difference, supra or Haber et al. , as stated in L22 Zahetoti Reinaly Record 84.1988 You can get it. The seed stock for virus purification cell culture is Freezing of master seed cell cultures followed by at least 19 post-infection passages That's what you get. Additional virus purification culture seed stocks are available at FIV Master Infection of continuous cat cell cultures with seed virus or originally FIV infected trout Single cell microwafer of high level FIV purifier from tarseed cell culture obtained by le cloning. For multiplication (breeding), master seed will Infected cat cell cultures were inoculated into cell tissue culture flasks. fused cells Following monolayer growth, tissue culture fluid was harvested at intervals of 2-5 days.

The seed virus works by killing master seed cells permanently infected with FIV. It is caused by proliferation due to cancer. The main material was added to the tissue culture flask.

This inoculum is in Relube Commodified Eagle Medium, which is Contains 2mML-glutamine and 4.5g/l glucose (DME), this DM E is 100 units/ml penicillin and streptomycin and 2mM glucose Contains tamin. The inoculum is added to a tissue culture flask, incubated, and treated. Rizumi's tissue culture fluid is used when cells grow into colonies. was taken on. Cells were separated from the culture vessels with trypsin/EDTA and :5 to 1:25 (typically 1:8). Preferably frass The water was kept constant at 36°C to 38°C for up to 7 days (3 to 7 days), then the fluid and Cells were harvested. The collected fluid (including cellular material) is placed in a high-speed centrifuge. (Sorva I RC-5B or Beckman J2-21) It was separated into supernatant and cell pellet. The resulting cervelet is discarded and the top Cleared culture fluid was used to obtain working virus. Separated (purified) The solution was 0.5M polyethylene glycol (PE0 800 0. Sigma) was set at 4% to 10% (usually 7%). Next, 2℃～ After being incubated overnight at 7℃, 1300XG (gravitational acceleration) for 30 minutes. The virus was pelleted by centrifugation and redispersed in the buffer. . Here, the buffer is 10mM Tris, pH 7, 6300mM NaCl, 2°C to 7°C with 1mM EDTA. After ordination night culture, the virus is 13 Centrifuged at 00xG for 115 minutes, the pellet was discarded and the supernatant was diluted with 10mM Trts in 300mM NaCl, 1mM EDTA at pH 7.6 The sample was centrifuged in a 50%/80% discontinuous glycerol step gradient. this Centrifugation was performed at 100,000×G and 4° C. for 3 hours. And 50% to 80 % interface FIV viral bands were collected. This band is 10 mMTris, 0.3M NaCl and 1mM EDTA were mixed and dispersed in the buffer. Diluted 3:3 in buffer and pelleted again by centrifugation at 100,000 x G for 1 hour. It was made into a computer. The pellet thus obtained is purified virus, and the pellet is the purified virus. Dispersion-mixed once more in buffer and stored at -70°C. thus obtained The virus is substantially free of FIV host cell proteins; At least 5% p26 (densitometric scan of -250 staining Measured as total white by SDS/PAGE Consists of cleocapsid protein). Such purified viruses are It can also be obtained by the following method. However, the applicant The polymeric contaminants present in I discovered that it can be removed by Here, high salinity means greater than physiological salt concentration. say something

As shown in Figure 1, it is associated with purified FIV. Polypeptides were analyzed by SDS/PAGE and infected using the same technique. Polypeptides uniquely isolated from treated culture medium of non-cells compared to de. Commass ie Blues stain gel According to analysis of stained GELS, three major polypeptides, namely The molecular weights are 10kD, 15kD, and 26kD, respectively, and plo, p15, and p26. The named polypeptide was found.

to the same cat with polyclonal antibodies against the FIV polypeptide. FL ISA test using FIV was carried out, and positive result was obtained by ELISA test. Western blot analysis was performed on feline lymph fluid (se r a) determined to be At times, each cat has a molecular weight of 10.15.26.4 under the conditions. had antibodies that reacted with polypeptides of 0 and/or 65 kD. .

As shown in Figure 2, 76 Proc of Tobin et al., Natl, Acad.

Standard Western described in Sc i USA 4350, 1979 Immunoplots were performed. FIV was ground with SDS and protein and nitro Transferred to cellulose sheet. Nitrocellulose sheet is 30% Bovine Lymph, 1% Bovine Lymph Albumin (BSA) and Dulbecco's Phosphate Contains 0.05% Tween 20 in buffer salts. This sheet is 0.5 Blocking buffer cut into cm strips and diluted 1:100 The cells were incubated for 2 hours at 20-22°C with the lymph samples inside. This strip is , washed repeatedly with wash buffer. This wash buffer is Dulbe cc.

0.05% Tween in phosphoric acid buffer salts. And with a second antibody cultivated. This second antibody combines the heavy chain and light chain of cat Ig. It specifies the This is also horseradish bar oxidase (CO Kirkguard and Perry Lab.

Obtained by Ratories Inc., Gathersburg, MD Ru. After 1 hour of incubation, the strips were washed repeatedly with wash buffer. So and incubated for 10 minutes with the aggregating substance 4-chloronaphthol. This strip is Partially dead, the results were immediately determined. Each lane A-G Lymph fluid was obtained from various cats infected with FTV. Dominant, Lothriak tivity was detected for p26 and p15, and to a lesser extent for plo.

Other proteins with molecular weights of 30, 40, 47 and 65 kD were also detected.

Certain viral polypeptides, such as gag (i.e. p26) antigen polypeptides, The peptide is found abundantly in purified virus preparations. virus envelope Other polypeptides, such as lobe polypeptides (e.g. gp130), tends to be lost during the refining of And compared to l1 main antigen, beranistan pro- The efficiency of electrical transfer is not good for head analysis. Therefore, the virus envelope (env) and gag precursor polypeptides for faster detection in FIV cells. The extract was labeled with 35S-methionine and cystenine, and Tested by Gnobresquitation (RIPA). infected with FIV Confuse culture of cells with methionine and cysteine Lee's Dulbecco's modified Eagle's medium This is done by incubation for 30 minutes in m. This cell culture was carried out in the same medium (me). dium) Srnl for 4 hours. Here, this medium is 358-methionine and 35S-cystenine of 100μ curie per rn1 (Inactivity 1200 curies/mM, New England Nuclear Corp. ration, Boston, MA), radioactivity marked in this way The tissue culture solution was removed and the cells were washed in 10mM sodium phosphate buffer, pH 7.5. Dissolved in 5 ml buffer. This buffer contains 100mM Na at pH 7.5. C1, 1% T "1 ton-X 100, 0.5% Sodium Deoxycholei g, 0.1% SDS, 0. 1. mM phenylmethylsulfonyl fluoride, and 100 kallikrein abrotiin activator units per ml. (100Ka l l l 1krn 1nactRvator units o f aprotenin per ml) (Sigma Chemical Company, Sen. (Truys, MO). Then, before use, the cell lysate was Normalization is achieved by centrifugation at 00XG for 30 minutes and the pellet is discarded. mark A portion of the identified cell lysate (0°1 ml) and 5 μm of lymph fluid to be tested were , mixed in a microcentrifuge tube and incubated for 1 hour at 37°C, then incubated at 4°C. It will be held overnight. The next day, 100mM NaCl, 1% Triton X-100 and 0. In 10mM phosphate buffer at pH 7.5 containing 1% SDS Protein A Sepharose CL-4B beads ( Pharmacy is also a biscuit way.

0.2 ml of a 5% floating suspension of NJ) was added to each tube and mixed for 30 min at 4°C. combined. Protein A bound to Sepharose beads The resulting antibody/antigen complex was collected by centrifugation (2 min, 20,000×G). and washed three times in lysis buffer. The final beret is again 2 Suspended in 5 μI SDS/PAGE loading buffer and incubated for 3 min at 10 Heated to 0°C. 5epharose beads are removed by centrifugation, The supernatant applied to PAGE Ge1s is newing to Enlightening TM. U.S. registered trademark of Rand Nuclear Corporation, Boston, MA ) and then applied to Kodak XR-5 film. Exposure at 70°C. Lymph from experimentally infected cats has been Recognizes peptides 15.22.36.40.47.11.0 and 130kD. death However, all cats are p22. For gp4o, gp47 and gp130 There are quantitative and qualitative differences in what appears to be a response. (mou nta response to p22・=) Regarding the main internal constituent protein p26 Which polypeptides are identified by the associated RIPA-PAGE analysis? To determine that, RI PA-PAGE analysis was performed using Western blocking It was performed using a monoclonal antibody that reacts with p26 as determined by. child Monoclonal immunoprecipitation of proteins p47, p36, p22 and p15. It shall be immunoprecipitated.

High molecular weight polypeptides (130 FIV of kD) was confirmed by RIPA-PAGA. Molecular weight 100kD protein was modified using lymph antibodies obtained from several infected cats. It was possible to According to another example, the antigenic glycopeptide of FIV is: You can get it as below. Actively growing CRFK FTLV infected cells was scraped from a roller bottle and soaked in phosphate-buffered salt (PBS). It was gently washed and made into a pellet. For cell pellets, 1 ml of buffer in pH 7, 2, 10 Mm sodium phosphate at a ratio of 0.1 ml of Cervelet to It was slowly redistributed. This suspension is heated on ice or in the refrigerator for 5 to 10 minutes. The temperature was maintained constant for minutes. Then, stir vigorously for 30 seconds to dissolve 4 volumes of PSB into IM. of PMSF. This mixture was prepared using a Brinkmann homogenizer-PT 10/35 (uses PTA20 generator) for 90-120 seconds It was homogenized.

After homogenization, it was purified by centrifugation at 5000xG for 20 seconds. Ta. The supernatant was discarded and the cell membrane pellet was rehydrated with PBS + 0.2%-Tr. iton X-100, buffer y2.5ml: 0.1ml original cell pellet It was then re-dispersed and floated at the same ratio. This mixture is again heated in a Brinkmann homogenizer. - PTIO/35 (PTA20 generator) provides strong power for 90 to 120 seconds. Modified. The obtained homogenized product was heated at 1.00000xG for 1 hour. The supernatant was removed with decanthisilane and the batch was purified with Pha rmac. i aLentil Lectin 5 epharose 4B on resin 6 ml to 5 ml of the original cell pellet, place at 20-23°C for 1 day. Landed (b o u n d).

This Lentil Lectin suspension is poured into the column. The resin was collected and added to 15 column volumes of PBS + 0.2% Trt. Washed with ton X-100. Glycoprotein contains 5 to 10 resins. Column volume of PBS + 0.2% Triton X-100 + 200mM methyl a -D Mannopyranosides (mannopyranos tde) The fat is eluted and a fraction of 1 column volume/tube is collected.

Separation of glycoproteins was confirmed by 9% 5O3-PAGE electrophoresis.

Then, test using 35S-radiolabeled cell preparations in combination with RIPA data. was checked.

Further purification of viral glycoproteins from host cell glycoproteins for HPLC and affinity chromatography. Including the use of polyclonal or monoclonal antibodies.

Example 1: gag polypeptide purification The isolated virus (250-1500 μl) was added to 2 volumes of 6M guanidine hydrochloride. , pH 3 (20% trifluoroacidica acidica in water) (prepared with cid)). This mixture was swirl mixed and 37 Place in water bath at ~40°C for 20-25 minutes. The solution obtained in this way is , pre-wetted (100 μl of 6M guanidine) Hydrochloric acid, pH 3) O145 Microgel Aqua Disilter (No. 4184) filtered by. And the filter is 100 μm 6M guanidine hydrochloride. Adjusted by pH3. The filtered sample was transferred to I (PLC column) for purification. loaded into RAM.

This PHLC system consists of three 110 v pump, 421 controller, 166 variable wavelength detector, 42 finch 210A injector with dynamic mixer and 1000μl sample. It has a sample. The column is equipped with a differentiated inde soto connector (water Microbond-A-back Zenile 10 Mo 8 mmX10c m Cartridge No. 85722 and Guard Pack Resolve CN cartridge 85826)) was held in an RCM-100 column holder with Waters Radial Compression Cartridge. This system The system is set so that the two levers are pressed together and the pressure is in the middle yellow zone. It was.

Purification was performed from 0.1% trifluoroacetic acid (V/V solvent A) to 0. ゜Acetonidrol containing 1% trifluoroacetic siad (V/V solution) By multi-stage gradient of medium B).

The fractions are freed from solvent to give a final volume of 50-100 μm. For this purpose, servant speed back (savant 5peed back) vac) is collected and concentrated. The concentrated fraction is 200-300μ By addition of 50mM or 100mM NaCl phosphate buffer at pH 7.2. It is considered neutral. The pH of the buffered fraction was checked using pH paper. If the pH is still below 6, add -normal NaOH and adjust the pH. to a range of 6.5 to 7.5. The neutralized fraction is used It is frozen at -20℃.

A print cut from the HPLC column described above is shown in FIG.

Starting with 100% of solvent A, the flow rate was 1 ml 17 min. After 15 minutes, solvent was changed to contain 26% solvent B. And after 100 minutes 31 solvent 811 37.5% solvent B after 2 minutes, 40% solvent B after 5 minutes, 43% solvent B after 6 minutes, 4 after 8 minutes 5% solvent B, changed to 60% after 155 minutes, and 10% solvent B after 200 minutes.

The peaks corresponding to plo, p15, p2'6 are marked in FIG. this Fractions containing these peaks were collected.

As shown in Figure 4, the fractions corresponding to plo, p15 and p26 are 5D S-polyacrylamide gel (containing 15% bis-acryl°7mide at 70V) ) It was inside. This gel is Biorad 5ilver Staining Silver staining was performed using Kit 161-0443. p15, plO and The fractions separated corresponding to FIV polypeptides and p26 are essentially FIV polypeptides. It is a homogeneous solution.

Isolated FIV polypeptides were sequenced using standard techniques for amino acid sequencing. The results are shown below. put it here,? is in the actual amino acid Indicates that something is not clear or that information about it is not available.

Ntnber l-2-3--5-8-7-8-9-1n-11-12-13- 14-1,5-16-17-18-19-20^1nO Acid P-toQ-T-V-N-G-V-P-Q-Y-V-A-L-D-P- Ni-M-V-3pl.

Nu+gber 1-2-3-4-5-6-7-8-9-10-11-12-1 3-14-15-16-17-18-19-^sln.

Acfd V-Q-8-R-G-8-G-P-V-C-F-N-C-ni-ni-P -G-H-L-Number　20-21-22-23-24^Afn.

Acfd ^-R-Q-3-H Number C1-C1-1-2-3-4-5-8-7-8-9-10-11 -12-13A.

Acfd 5-V-Q-8-R-G-Q-G-P-V-A-F-N-? this invention Whether or not the purified polypeptide is useful, i.e. Any standard method can be used to determine whether a compound is antigenic. can do. For example, an ELSA test can be performed. This test from feline lymph fluid to determine whether the polypeptide is cross-reactive. using a polyclonal antibody. The polypeptide may also be used with or without adjuvants. Can be injected into animals without auxiliary agents. Examples of animals include mice. , to see if antibodies are formed by this injection. These polypeptides are F Prevention of diseases caused by IV and methods to prevent FIV infection in cats Used for the production of chin. These vaccines are grown (produced) in the usual way. This is then injected into the cat by intravenous, subcutaneous, or other means. child When the level is 1-100μg/g-animal, the interval is The treatment lasts for 3 to 4 days and continues until FIV immunity is formed.

FIV monoclonal antibody Antibodies to FIV polypeptides can be used to confirm (identify) the purified polypeptides described above. useful and aids in the purification of polypeptides. This antibody is also suitable for any polypeptide. It is also useful for the determination of the antigenicity of Tid. For example, in the preparation of useful antibodies such as be. Such antibodies are useful for the detection and detection of independent or small numbers of FIV polypeptides. It is a monoclonal antibody that can be purified.

Ba1b/CJ (Jackson Labs) mice were prepared as described above. An initial injection of 50 Mg of FIV virus produced immunity. In addition, this Here, the FIV virus was 50 μg/mouse, and Difeo Bacto adjuvant are mixed one by one. After 12 hours, each mouse was given the supplement. 100 μg of FIV virus was injected intravenously without concomitant treatment. This increased injection Three days later, fusion was performed on mouse myeloma cell lines FO or p3X. 63-Ag8.653. Mitsudrog phase myeloma line fuge The specimens were collected on the day of the trial and checked for viability. Cells were rotated at 300G for 8 minutes. separated from the growth medium by processing and free of lymph [)M): (ser The mixture was resuspended in um free DME).

Mice inoculated with FIV by fusion were killed by neck dislocation. . Then, the lungs were removed aseptically. DME lungs do not contain lymph fluid (serum free DME) and 20ml of complete medium (C complete medium) (DME is 20% bovine Serum and 100 units of penicillin and streptomycin per iml A sterile veterinary container containing 1mM sodium pyruvate) It can be put on a plate. To release the cells, the lungs are passed through a 23-gauge needle. It was torn apart.

Cells were placed in a 50ml conical centrifuge tube and pelleted at 300G for 8 minutes. It was done. The whole knot was resuspended in 5 ml of 0.17 M ammonium chloride. , and placed on ice for 8 minutes. 5ml fetal bovine lymph (20%) bovine fet, al5erutn) was added and the cells were centrifuged at 300G for 8 minutes. It was once again made into a beret by processing. After being resuspended in 10 ml of DME , cells were counted, and lung and myeloma cells were mixed at a 3:1 ratio. Thin The cell mixture was pelleted by centrifugation for 200 GiO minutes. Kamisumi is big The pellets were placed vertically for 5 minutes to drain. or5min) and over 1 min], mm of 50% PEG (PEG 1500 mixed with 1=1 Pepes, pH 8.1) was added at 37°C. It was done.

After 1 min, kept at 37 °C, iml DME was added over the next 1 min period. Ta. Finally, 1.0 ml of DME was added over 2 minutes. The cells are 2 It was pelleted in 00G for 8 minutes. And Beret is a perfect medium (camp l The mixture was resuspended in ete medium). This perfect medium is 0.016 rnM thymidine and 0.1mM hypoxanthine e) and 0°5 μmo I/aminopterin and 1 -0% hybridoma cloning fac tor) (1, xHAT). The cells were plated in a 96-well plate. plated (plated).

3.5. After 7 days, remove the halves in fusion plates. The medium was removed and replaced with fresh 1x HAT. 11 days later, no\i Glidoma cell supernatants were screened by ELISA test. This te In the test, 9(')-we11 plates were incubated with FIV by standard techniques. Coated by Luz. 100 μl of supernatant from each well was Added to corresponding wells on cleaning plate and placed at 20-22°C for 1 hour. It was written.

Each well was then washed three times with distilled water and 100 μl of water was added to each well. horsesradish peroxy conjugate dide conjugate and boat anti-mouse (goat an ti-mouse) IgG (H+L)1. A, M (1:1500 dilu tion) was added to each well. It was then placed at 20-22°C for 1 hour.

After three washes with distilled water, the substrate OPD/hydrogen peroxide was added and incubated for 5-15 minutes. It was kept at constant temperature. 1.00μm stop solution (1M salt Acid) was added and the absorbance at 490 nm was detected. Optical density reading Cultures with a value of chondrophila barking were transferred to a 2 cm2 culture dish, and To this was added normal mouse pancreatic cells in 1,x HT medium. It's been 3 more days Afterwards, this entire 2 cm2 culture was screened once again for antibodies; If these tests are positive again, limiting diluent tiorb). Cells in each 2 cm2 culture were counted The concentration is adjusted by concentrating to 1×10” per iml. Cells were diluted with complete medium to contain 5.10 and 50 cells of hybridoma cells. Normal mouse pancreatic cells at a concentration of 1 rnl are added to this. The cell is 96-w el, plated on one plate and diluted respectively. 10 days later, flooring Plates are screened for growth. 37% of the wells showed growth. there was. Wells that showed positive growth were screened for antibodies and tested Wells that were positive were expanded into 2 cm2 medium and supplied with normal mouse pancreatic cells. Ta. The cloning procedure is repeated twice until stable antibody-producing hybridomas are obtained. repeated. Here, the cell culture is a 2-"9-79-75-15O culture. It spread into a container and ascites began to form here.

Due to the production of ascites, tristane (primed IRCF) mice was used. 0.5 ml of pristane is added to each The mice were injected intraperitoneally (IP) and the mice were allowed to rest for 10-60 days. this When 4.5X106 cells were inoculated into each mouse IP, the ascites was Formed for 14 days. Ascites was collected with a Pasteur pipette through a hole in the peritoneum. It was taken.

Glycoprotein antibodies can be similarly isolated and detected. In particular, molecular weight 40k Two glycoproteins: D (gp40) and 130kD (gpl, 30) Antibodies against are detected using PAGE and RIPA, respectively.

In this invention, monoclonal refers to the purification and immobilization of FIV polypeptide. It is of constant use. Here, the particular polypeptide is specific to FIV. Including those with sufficiently strong interactions with FIV shrimp talk and FrV antigens, For immunoassay tests, such as detection of FIV antigen by ELISA, etc. It is for use. To determine which of the monoclonal antibodies mentioned above are useful , one primary test was utilized. This is a monoclonal antibody or FIV antigen polyclonal binding to FIV. detected together with the null antibody conjugate (E LISA test).

Still other tests indicate that a monoclonal antibody has good resistance to one or more FIV antigens. Perform a Western blot to determine whether the test results are responsive or not. general In addition, monoclonals with weak reactivity appear as weak bands (fa) on Western plots. int bands) and are not suitable for this invention. Yet another test is Radioimmuno Recipe Teishigon Test (radioimmur+oprec+p ipatjon assay) (RIPA). In this RIPA, The FIV virus was labeled with 35S-methionine and a monoclonal antibody It reacts with and forms in the immunorecipitate. And imtubrecipitito works in 5DS-PAGE and uses autoradiograph to detect labeled proteins. Be roughed up. This analysis determines which monoclonal antibodies target precursor FIV antigen polypeptides. Is it possible to detect that the polypeptide is polypeptide and not just a mature polypeptide? The antigenic polypeptide described above is used to detect naturally occurring antibodies produced by cats. It is used to Such detection can be performed using standard immunoassay techniques. Ru.

For example, as described above, an ELISA test or the like is used. Test like this An example is shown below. This example is not intended to limit the invention and is not intended to limit the invention; Many other standard techniques can be used.

Example 2 - Antibody test A 96-well flat bottom microphone is required to conduct this test. Rotator strip (96well flat bottom m1crot iter atrips), and this strip contains the appropriate antigen to be tested (e.g. p26, p15 or plo).

The wells to be tested are 100111 solution containing 0.15 gg of antigen. 0.25 moles (mol, ar) of H7,5 in sodium citrate. well was covered with rose film and left overnight at 4°C. and tapped until dry. Antibodies were then added to o, 25 molar Overcoated by addition of 200 μm 1% BSA in sodium citrate. It was.

The wells were then placed at room temperature (20-25°C) for 1 hour and the wells were dried by tapping. be dried. 7.5% saccharide in 0.25 molar sodium citrate at 200 μm M-sugar was then added to each well and left at room temperature for 30 minutes.

The resulting strips can be used immediately or dried under vacuum at room temperature for 6 hours. , for later use.

The test is carried out using a 100 μm cat serum (please add the lymph fluid sample). It was held. Here, feline serum samples amp 1 e) is a positive control, negative control or Test serum (sera) l:10 in Dulbeccos PBS This PBS was diluted with 0.1% Bovine (bovine) serum. Albumin, 30% calf serum, and 0.05% Tw containing e-en-20 (Sigma Chemical, St. Louis, MO) in the wells. Served. The wells were then left at room temperature for 30 minutes to dry the wells by tapping. do. Wells were filled with Dulbecco-s PBS (0.05% Tween-2 0), and after this second cleaning, the The wells are then dried.

Next, 100 μl of a solution containing antibodies against feline immunoglobulin is added.

These antibodies are conjugated with indicator enzymes (e.g. alkaline phosphatase) ( cojuga t ad). and 50% calf fat lymph with pH 7.6. Contains fetal calf serum and 0.05% Tween-20. Dissolved in 0.05M Tris-HCl. Leave this well at room temperature for 30 minutes. dried by tapping and containing 0.05% Tween-20. Washed twice with Dulbecco's PBS.

3.0.1% in 40% glycerol and 50% methanol. 3”, 5.5 - A solution containing Tetramethylhensib is mixed in equal amounts with a solution containing the following: be done. That is, this solution contains 22.82 g of dibasic potassium phosphate in 11 , 19.2g of citric acid, and 1.34ml of 30% hydrogen peroxide solution. this 1.00 ml of solution is added to each well. and kept at room temperature for 15 minutes .

At the end of this constant temperature period, add 100 μl of 0.25% hydrofluoric acid to each wafer. Add to the bottle. Then, a wavelength of 650 nm was detected using a microtiter plate reader. The optical density is measured. ploSp15 and The immune response to p26 is Lymph fluid (feline 5era) was treated with both antibodies against FIV. It was detectable.

FiV nucleic acid FIV nucleic acids are useful for producing large quantities of FIV polypeptides. Also, FIV port It is also useful for producing fragments of lipeptides. Also, in vivo It is also effective for detecting homology of nucleic acids in vjvo. Furthermore, these In this case, the usual techniques are used. Next, the DNA of the FIV virus is An example of cloning will be explained below. Here, a specific FIV strain (s The selection of tra in) does not limit the invention. Those skilled in the art will The use of loaned sequences allows for the isolation of equivalent (substantially equivalent) nucleic acids. We recognize that the cloned sequences herein are specific to the specific deposit. Provided by. Equivalent nucleic acids can also be obtained using a method similar to the example described below. It is understood by those skilled in the art that the following can be obtained.

By FIV Strike 1 Twin 2428 (Pentaluma fsolate) A productively infected Crangal kidney cell line (Crangal ferine) kidney cell) as a base for unintegrated viral DNA. used. This unintegrated viral DNA is extracted by heat extraction (Hirte xtract1on). CsC1-Ethidium Digested into linear and supercoiled viral DNA by microbromide centrifugation. prepared (Hirt, 26 J Mo1. Biol, 365, 1967; C anaani et al., 2g2 Nature 378, 1.979).

Viral DNA sequences where supercoiled viral DNA overlaps It is used to build libraries containing. this A method for constructing such a library is described by Maguchi 1atis et al. C1oning, A Laboratory Manual, Co1d Spr ing Harbor Lab, Colorado Spring Harbor, N.

Y and Glover, DNA Cloning, Vol. 1. A Practi cal Approach, IRL Press, Washington, DC ) and is commonly used by those skilled in the art.

Supercoiled viral DNA is cleaved by two limiting endonucleases ( It recognizes the DNA sequence 5-GGCC3-) is a supercoiled wire. It is about the same DNA, and two sets with overlapping sequences. Generates blunt-ended DNA molecules. This smooth bundle 5--GAATCC-3” methylated adenine residues are limited endonucleases. The enzyme is modified to prevent cleavage of DNA treated with EcoRI. Methi The converted DNA molecule is bound to a linker (l1nker) DNA molecule (If The terminus of the generated molecule is then transferred to the recombinant DNA phage vector λZAP (U.S. Registered trademark: Stratagene, La Jolla, California) Compatible with the EcoRI cloning site. It's going to split open The linker fragment obtained by ger, Mannheim, Indianapolis, IN) f- size Separately, T4 DNA ligase (New Eng) is added to the AP vector. ligat (Land Biobabs, Beverly, MA) ed) to be done. The bound DNA molecules are made of Gigapack Gold (Stra, t agene.

(La, Jolla, California) into phages.

Various phages from this packaging reaction were collected from BB4 cells (Stratag ene) and plate lysates of these infected cells. The stock of recombinant λZAP clones is get the best results.

The insert DNA of the recombinant λZAP clone is in addition to the host cell DNA. Immediately detectable label with FIV proviral DNA sequence Screen each library with F IV DNA probe with It is necessary. Such probes are made from RNA isolated from FIV. will be built. Radioactive complementary DNA is Synthesized from natural viral RNA. For this RNA, Maniat It is described in fs et al. 1982. Here, poly A-c is not mentioned.

No selection of nttaintngRNA was performed, and Methymercuric/X Hydrogen The exception is that it is excluded from the protocol.

The bacteriophage library contains 10,000 bacteria in a 150 mrn dish. Plated at phage density. These are radio active (labeled nibrobe and nitrocellulose filter (Maniatis et al. 1989. Above et al. Fur immunoblized (tmmob L I L zed) on color 989 screened by hybridization with di-DNA. each hybrid Is (paired) bacteriophage plaques are suspended and Hybridization is performed in the same manner as described above. And one well was λZAP recombinant. Iterates until it contains a clone. XL-1-Blue cells (St ra t gene) is infected by recombinant λZAP phage. stop The plasmid containing the insert DNA is generated using the following R408 helper phage. Obtained by su7ku-inhuekungkong. Here, the production of helper phages is the manufacturer's guideline (Stratagene, La Jolla, California). by Niya). This technique uses a recombinant plasmid (which can be isolated from cells). ) and alt- strumput (st randed) phage stocks We also supply Here, this phage stock was used for DNA sequence analysis. can be separated from the medium.

The above-mentioned recombinant plasmid can be used for insertion using the prepared plasmid DNA. The data will be analyzed. This plasmid DNA is as follows. Sea urchin is purified by cell division in an overnight culture of bacteria. Ru. 1 or 1. ．． A 5 ml overnight culture is Placed in a microcentrifuge tube and spun at 4000G for 4 minutes. The skim is removed The tube is then spun again at 4000G for 4 minutes. The supernatant is also removed and The tube is rotated for several minutes. Then carefully remove the remaining liquid with a Pasteur pipette. removed by cut. Bacterial pellets were placed once again in a 200 μm solution. Distributed floating. This solution has a pI (8,05), 8% sub-potency and 50mm EDTA and 5% Triton X-100 and 50MM Tri Contains s/HC1. 1.0 mg/ml in 10 mM Trts/HCI 20 μl of irisome solution containing lysosomes and 1 μM EDTA were added and mixed. will be combined.

This mixture is then placed at 4° C. for 15 minutes. The solution containing this bacteria is , placed in a boiling water bath for 90 seconds. This mixture was cooled on ice and microfried. 1 at 11.000 G under cooled conditions in a microfuge. Rotated for 0 minutes. The pellet is carefully removed using a glass pipette. 2 00 μl of ice-cold isopropanol is added, the solution is mixed thoroughly, Place at -20°C for 5 minutes. The cooled solution was heated at -20℃ and 11000G for 10 minutes. The plasmid DNA is pelletized by centrifugation. Carefully remove the supernatant The pelletized DNA is partially air-dried. DNA Beret is Dissolved in 100 μm sterile double-distilled water. Plus separated in this way Mid-DNA is cleaved by limited enconuclease. analyzed for inserts. And agarose gel (Mania tis et al. 1982, supra). Electrophoresed in 8% gel.

Standard 2 deoxy sequence analysis is performed on recombinant DNA containing clones. It is carried out about. Single chain phage (single 5 tranded pHA GE) is separated from materials used for cell growth. This proliferated cell is Containing a Bluescript plasmid using the method described in the manual. I'm reading. That is, this method uses M13 dideoxy sequencing manifold. Bethesda Research Laboratories, Gaithersburg, Mary. There is a general description in the Rand) publication. The number of clones (n umb e r) is sequenced and analyzed using this method. l0CX, 2BY%R5X Sequence information about the regulated clones is shown in Figures 5aq, 5b, 5c. It is. What is shown in this Figure 5 is the estimated This is the amino acid sequence translated into . These amino acid sequences are: Shows homology to amino acid sequences such as In other words, it is immunologically close to FIV. Equine 1nfectious anbmia vfrUS), which has homology to the amino acid sequence of lentiviruses.

The nucleic acid probe obtained from the 2BY DNA sequence was infected with FIV. infected but not uninfected eelI hybridize (pair) with the DNA isolated from s). This probe is similar to other It can also be used to isolate other FIV genes from the strain. can be done. Alternatively, standard techniques for polypeptide purification as described above may be used. It can also be expressed.

Deposit Strains 10CX, 2B4 and R5X have been deposited with the ATCC and have received this The bars are 67937.67938 and 67939. Applicant and his designation fee In the event that this cultivation ceases before the expiry of this patent right term, the Confirm offering this again. This is the final requirement for culture shall be held responsible even after 5 years or 30 years or more. In addition, the issuance of such patents The bank is also responsible for informing the bank of the deposit location. The deposit will eventually be made available to the public. Until now, the deposit was 37 CFR1 14 and 35 U.S.C. Section 112 Deposit is available.

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Claims (13)

[Claims] 1. A purified polypeptide comprising an epitope of an antigenic polypeptide of FIV. Including. 2. 2. The purified polypeptide of claim 1, wherein the peptide is FIV contains epitopes of gag or env. 3. 3. The purified polypeptide of claim 2, wherein said polypeptide is A polypeptide having at least 75% homology to the gag or env polypeptide described above. Contains lipeptides. 4. 4. The purified polypeptide of claim 3, wherein said polypeptide is F. IV gag or env polypeptides. 5. 5. The purified polypeptide according to claim 2, 3 or 4, wherein the purified polypeptide p10, p15, or p26. 6. 5. The purified polypeptide according to claim 2, 3 or 4, wherein the purified polypeptide The peptide is gp40, gp100 or gp130. 7. A method for detecting antibodies against FIV, the method comprising: A step of purifying a polypeptide containing a pitope, and A step of contacting a sample containing the above antibody, and a step of contacting the sample containing the above polypeptide and the above antibody. detecting the presence of a complex formed between the two. 8. A purified nucleic acid containing 50 nucleotides naturally occurring in FIV particles. 50 nucleotide sequences with at least 90% homology to the otid sequence -includes 9. 9. The nucleic acid according to claim 8, wherein the nucleic acid is an antigenic polypeptide of FIV. Encodes a polypeptide containing an epitope. 10. 10. The nucleic acid according to claim 9, wherein the polypeptide comprises FlV gag or contains an epitope of the env polypeptide. 11. 9. The nucleic acid of claim 8, wherein the nucleic acid is present in an expression vector. 12. A method for purifying immunity against FIV-induced diseases in cats, comprising: inoculating a cat with the described polypeptide. 13. 10 or more purified polypeptides obtained from the following sequences: It has consecutive amino acids. [There is an array] or [There is an array] or [There is an array] Details of the invention explanation